A rapid, simple method for nudei isolation and purification from soybean (Glycine max L. Meff.) protoplasts is described. The isolated nudei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.Isolated nuclei have been used recently to investigate biochemical aspects in higher plants (2, 3). However, it is difficult to obtain adequate yields of intact nuclei in a short period of time from plant materials. This paper describes a rapid, simple procedure for isolation of intact nuclei from plant protoplasts. As criteria of the integrity and purity of nuclei, DNA analysis in CsCl density gradient centrifugation was done and the biological activity of the nuclei was ascertained by measuring amino acid incorporation, RNA and DNA synthesis. MATERIALS AND METHODSCell Culture and Preparation of Protoplasts. Cell material used in this work was soybean (Glycine max L. Merr.) (SB-1).The cells were maintained in suspension culture with 1-B5 medium (5). Protoplasts were prepared as described previously (7).Preparation of Nuclei from Protoplasts. Protoplasts prepared from 300 ml of SB-1 cell culture (approximately 13.5 ml packed volume after centrifugation at lOOg for 5 min) were washed with 0-B5 (sucrose-free) medium containing 0.275 M sorbitol and suspended to make a total volume of 20 ml of 0-B5 (sucrosefree) medium containing 10 mM MgCl2, 1 mm 2-mercaptoethanol, and 0.5% of Triton X-100 (medium A) plus 0.275 M sorbitol. The protoplasts were disrupted by a Dounce homogenizer with 10 gentle strokes. The homogenate was passed once through a layer of Miracloth and twice through triple layers of Miracloth. Filtrate was layered on 4 ml of medium A containing 0.4 M sorbitol and centrifuged at 400g for 5 min in an International model HN centrifuge or a Sorvall model GLC-2 centrifuge. The nuclei pellet was suspended in medium A containing 0.275 M sorbitol to make a total volume of 2 ml. This step was repeated twice to remove debris, cytoplasmic organelles, and starch granules. Nuclei suspension was layered on gradients of 5 ml of 0.5 M sorbitol in medium A and 2 ml of 1 M sorbitol in medium A, and centrifuged at lOOg for 3 min. The top layer, containing the nuclei, was withdrawn and spun down at 400g for 5 min and the pellet suspended in 2 ml of 0.275 M sorbitol in 1 NRCC No. 15964. medium A. This process was repeated at least twice to ensure complete removal of larger particles such as small unbroken protoplasts (checked under a light microscope). The nuclei were suspended in 1 to 2 ml of 0.01 M tris-HCI buffer (pH 7.5) containing 0.275 M sorbitol, 10 mM MgC92, 1 mm 2-mercaptoethanol, and 0.1% of Triton X-100 (Fig. 1), and used for assaying amino acid incorporation, RNA and DNA synthesis and for DNA isolation and analysis. Nuclei number was determine...
DNA binding and uptake by ucdei isolated from soybean (Glycine max L. Meff.) protopbsb were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drsicay with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occumd at pH 6 and the procea was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dTJ-[dA-dTJ) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-L-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.Genetic modification of higher plants by feeding exogenous DNA has been reported (4,8,14). Because plant protoplasts lack a cell wall they may have distinct advantages over normal plant cells for exogenous DNA uptake (5,11,13,15). Incorporated DNA may be degraded and reutilized for de novo DNA synthesis prior to genetic expression. Exogenous DNA must be stabilized as well as replicated before the genetic information can be expressed in the cell. A possible mechanism for stabilization is the integration of DNA into host genome. Exogenous DNA must therefore penetrate through the cytoplasm, and must be adsorbed to nuclear membrane without being degraded.Some investigators have observed that DNA taken up by cells was bound to the nuclei using radioautographic techniques (6, 7) and by analyzing nuclei isolated from protoplasts fed labeled DNA (15). This paper describes some of the factors affecting in vitro DNA binding and DNA uptake by nuclei isolated from soybean protoplasts. MATERIALS AND METHODSCell Culture and Preparation of Protoplasts. Soybean cells (Glycine max L. Merr.) (SB-1) grown in 1-B5 medium (3) were used throughout this work. Protoplasts were prepared by enzymic removal of cell wall as described previously (10).Isolation of Nuclei from Protoplasts. Protoplasts washed with 0-B5 medium (sucrose-free) containing 0.275 M sorbitol were suspended to 0-B5 (sucrose-free) medium containing 10 mm MgCl2, 1 mm 2-mercaptoethanol, and 0.5% Triton X-100 (medium A) plus 0.275 M sorbitol. The protoplasts were disrupted by a Dounce homogenizer with 10 gentle strokes. The homoge-NRCC No. 15963. 98 nate was passed once through a layer of Miracloth and twice through a triple layer of Miracloth. Filtrate was layered on 4 ml of medium A containing 0.4 M sorbitol and centrifuged at 400g for 5 min in an International model HN centrifuge. The pellet was suspended in 2 ml of medium A containing 0.275 M sorbitol. This step was repeated twice to remove debris, cytoplasmic organelles, and starch granules. The nuclei suspension was layered on stepwise gradients of 5 ml of 0.5 M sorbitol in medium A and 2 ml of 1 M sorbitol i...
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